New homozygous gpt delta transgenic rat strain improves an efficiency of the in vivo mutagenicity assay

Abstract

Background: Gene mutation assays in transgenic rodents are useful tools to investigate in vivo mutagenicity in a target tissue. Using a lambda EG10 transgene containing reporter genes, gpt delta transgenic mice and rats have been developed to detect point mutations and deletions. The transgene is integrated in the genome and can be rescued through an in vitro packaging reaction. However, the packaging efficiency is lower in gpt delta rats than in mice, because of the transgene in gpt delta rats being heterozygous and in low copy number. To improve the packaging efficiency, we herein describe a newly developed homozygous gpt delta rat strain.

Results: The new gpt delta rat has a Wistar Hannover background and has been successfully maintained as homozygous for the transgene. The packaging efficiency in the liver was 4 to 8 times higher than that of existing heterozygous F344 gpt delta rats. The frequency of gpt point mutations significantly increased in the liver and bone marrow of N-nitroso-N-ethylurea (ENU)- and benzo[a]pyrene (BaP)-treated rats. Spi^- deletion frequencies significantly increased in the liver and bone marrow of BaP-treated rats but not in ENU-treated rats. Whole genome sequencing analysis identified ≥ 30 copies of lambda EG10 transgenes integrated in rat chromosome 1.

Conclusions: The new homozygous gpt delta rat strain showed a higher packaging efficiency, and could be useful for in vivo gene mutation assays in rats.

Keywords: Spi− assay; gpt assay; gpt delta transgenic rat; mutant frequency; mutation spectrum.

Fig. 1

Genomic integration and homozygosity of EG10 transgene. Genomic integration of transgene was confirmed by multiplex PCR using three primers. Primer sequences are presented in Supplementary Fig. 1. Genomic DNA extracted from liver of WH homozygous gpt delta rat line 2 and wild-type rat was used as a PCR template. A diagram shows primer positions for each genotype.

Fig. 2

Packaging efficiencies of homozygous gpt delta rats. Two candidate WH homozygous gpt delta rat lines were analyzed for packaging efficiency. F344 heterozygous gpt delta rats were used as a control. Three animals were used in each group. Genomic DNA was extracted from the liver of untreated rats. Three independent in vitro packaging reactions using 10 µL DNA were conducted for each DNA sample. E. coli C and YG6020 cells were infected with the packaged phages. The number of rescued phages per packaging reaction (plaque forming unit: p.f.u or colony forming unit: c.f.u) was estimated. Error bar represents standard deviation

Fig. 3

The gpt mutant frequencies in the liver and bone marrow of BaP- or ENU-treated homozygous gpt delta rats. Error bar represents standard deviation. * P < 0.05, significantly different from vehicle control (Steel test). # P < 0.05, significantly different between male and female (t-test)

Fig. 4

The Spi⁻ mutant frequencies in the liver and bone marrow of BaP- or ENU- treated homozygous gpt delta rats. Error bar represents standard deviation. * P < 0.05, significantly different from vehicle control (Steel test). # P < 0.05, significantly different from vehicle control (Dunnett’s test)

Fig. 5

Integration of lambda EG10 transgenes in chromosome 1 of homozygous gpt delta rats. A diagram of the insertion region of EG10 sequences in the gpt delta rat genome is shown. The dark arrow represents the lambda EG10 transgene copies, and the white arrow represents the rat chromosome. Deletion (755 bps), inversion (699 bps) and small Indels (16 bps and 1 bp insertions, 9 bps deletion) in the rat genome were detected at the junction